Transcription factors play an essential role in organism development and homeostasis; and loss or impaired function of transcription factors has been implicated in a variety of developmental disorders and chronic diseases. Here, we show the transcription factor Ras Responsive Element Binding Protein 1 (RREB1) is essential for Purkinje cell survival in the mammalian brain. We identified a spontaneous mutation that results in hypomorphic loss of Rreb1 and leads to progressive Purkinje cell degeneration and ataxia in mice. ChIP-seq of RREB1 in wild-type cells and RNA-seq in wild-type and Rreb1-deficient Purkinje cells demonstrated RREB1 directly, positively regulates genes associated with the microtubule network and autophagy.Dysregulation of genes associated with the microtubule network lead to alterations in posttranslational modifications of tubulin and reduced dendritic branching in Purkinje cells. Altered expression of genes associated with autophagy lead to a reduction in autophagosomes and lysosomes, and the accumulation of P62- and Ubiquitin-positive inclusions. Interestingly, while this study demonstrates a role for RREB1 in the microtubule network and proteostasis in neurons, examination of RREB1 in HEK 293-T cells suggests it plays a different role in this cell type. Analysis of RREB1 ChIP-seq data from Purkinje cells and HEK 293-T cells identified an overlap in many target genes. Surprisingly, while RREB1 positively regulates these genes in Purkinje cells, it appears to negatively regulate these genes in HEK 293-T cells. Examination of autophagic flux in Rreb1 knockout cells demonstrated no significant difference between knockout cells and wild-type cells. Together, these studies demonstrate a novel and specific role for RREB1 in maintaining the microtubule network and proteostasis in mammalian neurons.

Focal Malformations of Cortical Development (FMCD) are neurological developmental disorders that are a major cause of drug-resistant pediatric epilepsy. Along with seizures, patients display disrupted cortical architecture and neuronal organization. Current treatment involves invasive surgical resection of the epileptic focus, which may only show partial benefit. Recent studies have identified several categories of post-zygotic somatic mutations that cause FMCD, however this effort has not yet been utilized to generate therapies. Antisense oligonucleotides (ASOs) are safe, stable and programmable molecules that can be designed to target specific mutations such as those that cause FMCD. To demonstrate the therapeutic potential of ASOs against FMCD, we introduced AKT3E17K, an FMCD causing variant in mice brain and subsequently treated them with ASOs designed to silence the toxic gene. The ASO treatment was able to rescue cortical architecture. Later studies may explore the dependency of treatment on the stage of the disease, the dose of the drug, and the type of FMCD causing mutations. Successful development of ASO therapy can provide safe, effective and non-invasive treatment for FMCD.

Neuropeptides serve as important neurotransmitters in the nervous system, essential for cell-cell communication among neurons performing diverse functions. This thesis aims to summarize, analyze, and provide further insight into the results of the Podvin et al. (2022) study, which applied methods from the Jiang et al. (2021) paper, and the implications that the results may have in the field for identifying therapeutic targets. The Jiang et al. (2021) study identified unique neuropeptides generated from differing proteases in dense core secretory vesicles (DCSVs) at a pH of 5.5 compared to the extracellular pH of 7.2. The production of distinct neuropeptides in purified secretory vesicles isolated from the bovine adrenal medulla (also known as chromaffin granules) involved aspartic, cysteine, serine, and metallo proteases. Through the use of neuropeptidomics, proteomics, and substrate profiling, the study by Podvin et al. (2022) demonstrated significantly dysregulated neuropeptides, proteins, proteases, and flanking residues of neuropeptides with proneuropeptide precursors between AD and aged-matched control synaptosomes. Additionally, through an extensive review conducted on the results of the Podvin et al. (2022) study on the proneuropeptides and proteases found to be dysregulated between AD and age-matched control, they were explored for their roles in either protection from AD pathology or as causative agents. The results from these studies indicate that combining proteomics, neuropeptidomics, and substrate profiling may be of great importance in exploring different disease pathogenesis to understand the dysregulation occurring in AD and for exploring new targets for drug therapies.

Charcot-Marie-Tooth type 2B (CMT2B) is a debilitating hereditary peripheral sensory neuropathy. Patients with this autosomal dominant disease lose pain sensation and experience muscle weakness and atrophy in distal limbs. Clinical manifestations of CMT2B include axonal dysfunction and degeneration of peripheral sensory neurons. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M) in Rab7, a regulator of late endocytic transport. Strong evidence suggests that CMT2B mutations enhance the cellular levels of activated Rab7 GTPase, thereby increasing lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor and epidermal growth factor in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A “gain of toxicity” model has, thus, been proposed based on these observations. In order to test the hypothesis that CMT2B mutations cause neural degeneration in a gene dosage-dependent, gain of toxicity manner, this study compares cutaneous autonomic innervation in footpad biopsies of wild-type, heterozygous and homozygous CMT2B mouse models. The results show significant neuronal degeneration associated with both disease genotypes compared to the wild-type control. In addition, disease phenotypes present in a gene dosage-dependent manner, with sensory deficits severely worsened in the homozygous model compared to the heterozygote. These results are consistent with data from previous behavioral tests showing a similar dosage-dependent effect of CMT2B mutant genes. Together, these findings affirm the gain of toxicity model of the disease and suggest that therapeutic development should aim to inhibit Rab7 hyperactivity in affected patients.

Errors in tRNA aminoacylation can lead to misfolded proteins, which can form protein aggregates. Protein aggregation have been linked to many neurological diseases, thus understanding the mechanisms that can lead to the formation of protein aggregates is critical for researching degenerative illnesses. Aminoacyl tRNA synthetases (AARSs) have been found to have hydrolytic editing functions that can correct tRNA mischarging errors and prevent mistranslation. A mutation, referred to as sticky, has been discovered to cause a dysfunction in the hydrolytic editing function of alanyl-tRNA synthetase (AlaRS), resulting in mischarged Ser-tRNAAla and causes serine to be incorrectly incorporated into newly-formed peptides. However, Ankrd16, a newly discovered protein, has been found to aid AlaRS in editing of mischarged tRNAs by removing serine from the aminoacyl active site of AlaRS. By conditionally deleting Ankrd16 in CaMKIIa-positive neurons in the forebrain of Aars^sti/sti mice, the Ackerman lab was able to induce neuronal death in targeted neurons (Vo et al., 2018). In this study, we attempted to induce motor neuron death by conditionally deleting Ankrd16 in ChAT-positive motor neurons in Aars^sti/sti mice. Although motor neuron death was not observed, other phenotypes, such as decreased weight, kyphosis, and decreased axon diameters were found to be caused by the conditional deletion of Ankrd16. These results suggest that motor neurons may be more resistant to Ankrd16 loss than other neurons, such as Purkinje cells.

Huntington’s disease is a debilitating autosomal dominant neurological condition which causes neural degradation and psychological and motor degradation depending on the severity of the expansion of a trinucleotide repeat responsible for its symptoms. Studies have found that longer expansions of the trinucleotide repeat correlate with earlier onset of the symptoms of HD; with very long expansions, the symptoms can appear as early as childhood. Symptoms of HD include conditions such as motor and cognitive dysfunction, loss of daily living capacity, weight loss, increased risk of death, whose severity have been found to be associated with the length of the CAG trinucleotide expansion. HD also causes repeat-number associated physical degeneration of brain tissue in areas such as the cortex, putamen, striatum, and various others. While many studies have been conducted on JHD in animal and cell models, few studies have analyzed the disruption to the proteome in human JHD brains. In this project, we determined the number of CAG trinucleotide repeat numbers and investigated the dysregulation of the proteome in the BA4, BA6 cortex and putamen of JHD brains compared to age-matched control brains, as well as curating a database of Htt-interacting proteins from animal studies which allowed us to determine which of the dysregulated proteins are Htt-interacting and are likely the first suspects in the many protein network disruptions observed in JHD. We found that there are significant protein network disruptions in the mitochondria, translation and RNA processing, and cellular transport functions, as well as various others.